Several possible mechanisms capable of inducing the anti-bronchoconstrictor effects of eprozinol have been investigated in vitro by means of the following techniques: (l)test for inhibition of mast cell histamine release, secondary to a passive anaphylactic response on human lung tissue; (2) determination of the type of interference with the contracting effects of bradykinin, acetylcholine and serotonin; (3) interference with the smooth muscle contraction induced by BaCl2 or CaCl2; (4) investigation of blockade by propranolol of eprozinol-induced relaxation of tracheal muscle; (5) investigation of changes in cAMP and cGMP levels in guinea pig trachea; (6) investigation of an inhibitory effect on phosphodiesterase. When the immunological reaction is carried out while eprozinol is in contact with the lung preparation, the compound appears more effective than disodium cromoglycate but less effective than isoprenaline in inhibiting mast cell degranulation. In fact, a 40% inhibition of mast cell histamine release occurs with a concentration of eprozinol at 2 x 10–6M. On the other hand, when the preparations are rinsed between the period of incubation with the compound and the addition of antigen, only disodium cromoglycate still manifests significant inhibitory activity. Eprozinol antagonises serotonin, bradykinin and acetylcholine in a non-competitive fashion (pD2 = 4.21 ± 0.09; 3.81 ± 0.07 and 4.17 ± 0.07, respectively). Eprozinol non-specifically antagonises BaCl2 (ileum or tracheal muscle from the guinea pig) and CaCl2 (KC1-depolarised guinea pig caecum). The relaxant effects on calf tracheal smooth muscle are characterised by the following parameters: isoprenaline, pD2 = 7.93 ± 0.21 and eprozinol, pD2 = 4.37 ± 0.17. With guinea pig trachea, these parameters are: isoprenaline, pD2 = 6.7 ± 0.10 and eprozinol, pD2 = 2.5 ± 0.09. In all cases the intrinsic activity of both compounds is the same (α = 1). No significant changes in cAMP and cGMP levels are observed in guinea pig trachea, with eprozinol or isoprenaline, at doses capable of inducing relaxation. Eprozinol is only a very weak phosphodiesterase inhibitor, at large concentrations.
The role of IgG4 antibodies in allergic disorders is suspected. Yet, their presence on human basophil membrane has not been demonstrated and the mechanism of the degranulation induced by anti-IgG4 antibodies remains unclear. As previously reported, we observed that monoclonal anti-IgG4 (10 to 100 micrograms/ml) induced histamine release in the presence of D2O from leukocytes of normal and atopic subjects. The release was accompanied by a decrease of the number of toluidine blue-positive basophils (TB+). Histamine release and TB+ decrease were also observed with lower concentrations of anti-IgG4 (1 to 100 pg/ml). Since basophil activation assessed by TB+ decrease was more sensitive than histamine release, we thus used the former method to further study the mechanisms of the anti-IgG4- vs anti-IgE-induced basophil activation. Basophil activation by anti-IgG4 at 1 to 100 pg/ml, but not by anti-IgG4 at 10 to 100 micrograms/ml or anti-IgE, required the presence of polymorphonuclear cells. Furthermore, anti-IgG4-stimulated purified eosinophils, but not neutrophils, released basophil-activating factors identified as cationic proteins from eosinophils. Thus, the human basophil can be activated by anti-IgG4 via two different mechanisms according to the antibody concentration. At high concentrations (10 to 100 micrograms/ml) basophil activation does not require the presence of polymorphonuclear cells whereas at lower concentrations (1 to 100 pg/ml) the presence of eosinophils is necessary. We propose that in the latter concentration range, basophil activation is a two-step process: 1) release by anti-IgG4 of eosinophil cationic proteins that 2) will, in turn, activate human basophils. This study lends support to the role of IgG4 and eosinophils in anaphylactic reactions.
SUMMARY The release of mediators from human basophils is strongly enhanced by lL-3. However, the signalling pathways of IL-3 arc poorly defined in these cells. Since external Ca2+ and Na+ play important regulating roles in histamine release, the possibility that these cations could be involved in the potentiation by rL-3 of the anti-IgE-induced histamine release from human basophils was considered, and it was observed that: (i) lL-3 dramatically decreased the external Ca2+ requirement for IgE-mediated histamine release. However, histamine release from IL-3-treated basophils became only partially independent of external Ca2+, since addition of EGTA in the external medium abolished the effect of IL-3; (ii) decreasing Na+ influx by lowering external Na+ concentration in isosmotic medium inhibited thepotenEiatiiigefTeLtoriL-3on IgE-mediated release; (iii) amiloride. An inhibitor of Na+/Ca2+ and Na+/H+ exchanges, and its derivative, benzamil, more specific for Na+/Ca2+ exchanges, inhibited the release potentiated by IL-3. In contrast, the amiloride derivative 5-(N, N-dimethy1)-amiloride more specific for Na+/H+ exchanges, slightly increased the IL-3-enhaneed release. Thus, the decreased requirement for external Ca2+ and the dependence on external Na + taken with the effect of the Na+/Ca2+ exchange inhibitors, suggest that Na+/Ca2+ exchanges are involved in the IL-3-indueed enhancement of IgE-mediated human basophil histamine release.
